Colorectal Cancer Screening: A Comprehensive Review to Recent Non-Invasive Methods.

Colorectal cancer (CRC) is one of the most common cancers worldwide and considered to be one of the hassles in medical communities. CRC develops from precancerous polyps in the colon or rectum and is preventable and curable by an early diagnosis and with the removal of premalignant polyps. In recent years, scientists have looked for inexpensive and safe ways to detect CRC in its earliest stages. Strong evidence shows that screening for CRC is a crucial way to reduce the incidence and mortality of this devastating disease. The main purpose for screening is to detect cancer or pre-cancer signs in all asymptomatic patients. In this review, we holistically introduce major pathways involved in the initiation and progression of colorectal tumorgenesis, which mainly includes chromosome instability (CIN), microsatellite instability (MSI), the CpG island methylator phenotype (CIMP), and we then will discuss different screening tests and especially the latest non-invasive fecal screening test kits for the detection of CRC.


INTRODUCTION
About a quarter of all deaths in countries with a westernized lifestyle are caused by cancer 1 . Colorectal cancer (CRC) is the third most common cancer among men and the second among women, and it is the fourth leading cause of cancer-related mortality worldwide 2 .In 2017, there will be diagnosed 95,520 and 39,910 new cases of colon and rectal cancer in the US, respectively 3 . In the last few decades, the incidence of CRC has been rapidly increased in Asia 4 . Approximately, 5-6% of the Western population will suffer from CRC during their lifetime 2,5 . Obesity, a diet low in fruit and vegetable, physical inactivity, smoking and a sedentary lifestyle are risk factors for CRC 6,8 . In this regard, lack of physical activity has shown to have a strong effect in the development of CRC by reducing the risk 25% 9 . Long-term treatment with aspirin, a low-fiber and Mediterranean diet may prevent colorectal cancer as well 7,10,11 . Colorectal cancer is classified into three major forms: sporadic, hereditary and familial. About 75-80% of CRCs are sporadic type in which somatic mutations are frequently found and are not associated with family history 12 . Hereditary colorectal cancer, Lynch syndrome and Familial adenomatous polyposis (FAP) account for 10% of all cases of CRC 13 . Several studies estimated that approximately 25% of all CRC cases are familial and do not follow the classical Mendelian inheritance pattern 14,15 . Over International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir the past few years, scientists have found that CRC is a heterogeneous cancer and hard to be diagnosed and treated through identification of its molecular and genetic characteristics 16 . The screening programs have declined the incidence rate of colorectal cancer by 4.3 percent per year among people 50 and above, but the CRC incidence rate increased by 1.8 percent per year among people under 50 17,18 , necessitating consideration of ages for screenings. Table 1 shows the classification of CRC. At least, four genetic and epigenetic mechanisms have been explained in CRC: 1) chromosomal instability (CIN); 2) microsatellite instability (MSI); 3) the CpG island methylator phenotype (CIMP) and 4) other mechanisms including inflammation and microRNAs. In this review, we will holistically explain the mentioned mechanisms and will bring up eminent methodologies for the current screenings and detection of CRC.
Genetic abnormalities implicated in the chromosomal instability pathway More than 80 somatic mutations have been identified in CRC by sequencing, only a few number of these mutations are significantly associated with CRC 19 .

WNT signaling components
Initial genetic change in sporadic colon cancer and FAP (familial adenomatous polyposis) tumorgenesisisan activation of Wnt pathway and abnormalities in chromosome 5q. WNT ligands belong to a large family of proteins that play very important role in the development of normal cells. WNT binds to the membrane receptors and triggers signaling cascade which is involved in an important process of embryonic development and adult cell homeostasis such as cell differentiation, cell polarity, and cell death 13 . Wnt pathways are divided into two common categories: canonical (β-catenin dependent) and non-canonical (independent of βcatenin) Wnt signaling pathways 4,20,21 . About 90% of sporadic colon cancers carry mutations in the WNT pathway 22 . APC gene, a tumor suppressor gene, has 15 exons and is located on chromosome 15q. APC proteins bind to β-catenin and are main components in the destruction complex. The APC mutations cause a truncated product with an abnormal function 22,23 . Beta-catenin is normally found in the cell membrane, but in the absence of APC, it is usually accumulated in the nucleus 24 . Germline mutations in the APC gene are responsible for familial adenomatous polyposis (FAP), however, somatic mutations in APC occur in 80% of sporadic colorectal tumors. A familial colorectal cancer syndrome such as FAP with an autosomal dominant inheritance is characterized by the development of hundreds or thousands of adenomas in the colon and rectum; the average age at FAP is 39 years 25 . Attenuated FAP (AFAP) is characterized by the presence of less than 100 adenomatous polyps; the germline mutations occur in 5′ and 3′ of the APC gene. MYH-associated polyposis (MAP) is caused by mutations in the mutY homolog (MYH) gene. MAP is inherited in an autosomal recessive manner, and thus individuals with MAP have biallelic MYH mutations. These patients often have no family history of colon cancer or polyps in their parents (although siblings may be affected). MAP and AFAP are often phenotypically similar 26 .
Aneuploidy: 18q loss DCC, SMAD2 and SMAD4 genes are all located on 18q and the loss of an allele accounts for 60% of CRC, and it is associated with a poor prognosis in stage II and III of CRC 27 . DCC gene plays important roles in the regulation of cell adhesion and migration and stimulates cell death in the absence of its ligand (netrin-1). Smad proteins are transcription factors that are involved in the  34 . More than 90% of the mutations in the K-RAS gene happen at codon 12 and 13 (35). Several studies have demonstrated that K-RAS mutations are associated with a poor prognosis in aggressive CRC and are predisposing factors for CRC metastasis to liver 36,37 .

Tp53 gene
Tp53 gene is a tumor-suppressor gene with 12 exons and 11 introns which is located on chromosome 17p 38 . Its mutations are one of the main steps in colorectal carcinogenesis. About 80% of TP53 mutations are missense mutations. As a tumor suppressor, Tp53 has different roles including the ability to induce cell cycle arrest, DNA repair, senescence, and apoptosis 39 . Furthermore, it has a number of transcription independent cellular activities essential for the maintenance of genomic stability. TP53 mutation is observed in about half of all colorectal cancer cases 40,41 .

Inflammatory pathway
One of the critical components in the CRC initiation and progression is chronic inflammation. Chan et al. examined the influence of C-reactive protein (CRP), Interleukin-6 (IL-6) and soluble tumor necrosis factor receptor 2 (sTNFR-2, a TNF-α receptor superfamily member) in CRC in a cohort of 33,000 women. Their results demonstrated an increased risk of CRC in women with high levels of sTNFR-2, but no association with other two markers was found. Remarkably, women with high baseline levels of sTNFR-2 who took aspirin had a lower risk of developing CRC 55,56 . Therefore, inflammation is an important contributor to colorectal carcinogenesis, and thusanti-inflammatory drugs have a protective effect on CRC 57 .

MicroRNAs
MicroRNAs (miRNAs) are a group of small noncoding RNAs which contain 18-24 nucleotides and regulate protein expression mostly by inhibiting mRNA translation of genes involved in cell differentiation, development, proliferation and apoptosis 58 . Recently, scientists have discovered that miRNAs are associated with CRC pathogenesis 59 . For instance, it has been reported that miR-145 and miR-143 are usually downregulated in precancerous adenomas compared to normal tissue 60 .
In several studies, scientists found a downregulation of miR-143 and -145 in stool samples of CRCs as compared to healthy controls 61,62 . Another study showed miR-144 to be upregulated in stool samples of CRC patients. Sensitivity and specificity of CRC detection were 74% and 87%, respectively 63 . Therefore, it has been suggested that dysregulated miRNAs may be useful markers for early detection or follow-up of CRC patients 60 . Therefore, miRNAs have great promises for the detection of precancerous adenomas and are being broadly studied for screening purposes in CRC.

Colorectal cancer screening
In recent years scientists have investigated new methods for a rapid detection of CRC that are less expensive, non-invasive and also have an appropriate sensitivity and specificity. Since 1985, death rate from CRC has been reduced because of early detection, and much of the reduction was due to the screening of people aged 50 to 75. There are a variety of methods and tests for the detection of CRC such as colonoscopy, sigmoidoscopy, fecal occult blood test (FOBT), fecal immunochemical test (FIT), double contrast barium enema (DCBE) and computerized tomography (CT) scan 64,65 . According to the American Cancer Society, if CRC is diagnosed at an early stage, the survival rate is more than 90%. In recent years, improvements have been made with the stool DNA testing as non-invasive and inexpensive tests for the diagnosis of CRC 66 .
International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir

CRC screening tests
As mentioned above, five screening tests are traditionally used for colorectal cancer: fecal occult blood test (FOBT), sigmoidoscopy, colonoscopy, barium enema and digital rectal exam. Moreover, some new screening tests have recently been studied: stool DNA testing and computerized tomographic colonography (CTC). In the following section, we will introduce these tests and their advantages.

Flexible sigmoidoscopy
More than 80% of CRC patients will be diagnosed if the left colon and rectum are examined by sigmoidoscopy. The purpose of this inexpensive method is to identify adenomas in patients between the ages 55-65 67,68 .

Colonoscopy
Colonoscopy is the gold standard method for CRC screening. Using colonoscopy, we are able to examine the entire colon and rectum, but this method has some disadvantages such as being expensive, needing a bowel preparation beforehand and having a risk for rupture 69 .

Barium enema
Like colonoscopy, this method examines the entire colon and rectum. It is cheaper than colonoscopy. However, the intestine needs to be prepared beforehand and is an invasive procedure 70 . Moreover, the barium enema fails in detecting flat or < 5 mm lesions, and also polypectomy or biopsy is not possible ( Table 3).

Computerized tomography
Computerized tomography (CT) is a new method for screening. This method is cost-effective and affordable in 5 minutes with similar sensitivity to colonoscopy and barium enema 69 . However, there are significant limitations in CT: first, the colon should be thoroughly clean; secondly, polypectomy is not possible. In addition, flat lesions are missed 71 .

FOBT
This test detects occult blood (hemoglobin enzymatically) in the upper and lower digestive tract 72 .
However, several studies have demonstrated its limited sensitivity for advanced adenomas (11%) and cancer (13%). FOBT is a noninvasive method and if a positive result is found, a colonoscopy is then recommended. However, approximately 13% to 42% of positive FOBT cases have negative colonoscopy 73 . A systematic review showed that the sensitivity of FOBT for CRC is 51% to 100%, while its specificity is 90% to 97% which will be higher if the test is repeated annually or biennial 74,75 .

Fecal immunochemical test
Fecal immunochemical test (FIT) detects human globin with a specific antibody (76). The sensitivity and specificity of this test are higher than FOBT (77,78). FIT Specificity for the prediction of colorectal cancer and adenoma is 90.1% and 90.6%, respectively, and the sensitivity of this method for neoplasms in the colorectal cancer and adenoma are 92.3% and 33.9%, respectively 78 .

Colosure™ test kit
Recently, scientists discovered that the Vimentin gene is hypermethylated by 53-84% in colorectal cancer. Closure™ test kit (Laboratory Corporation of America, http://www.labcorp.com) identifies methylation in the Vimentin gene. The exact performance of this epigenetic marker in detecting advanced adenomas is largely unknown. However, the Colosure kit is the only commercial kit that is available for screening clinical colorectal cancer in the US. The sensitivity and specificity of this kit for colon cancer is 72.5-83% and 53-86.9 %, respectively (Table 4).  (111) KRAS is a guanosine triphosphate/guanosinediphosphate (GTP/GDP)-binding protein and is widely expressed in various human cells. As a GTPase protein, KRAS is involved in intracellular signal transduction and mainly responsible for EGFR-signaling activation (112) 12p12.1 KRAS Gene ACTB is considered as a housekeeping gene so that its expression is not usually affected by changing conditions; therefore it is widely used as internal control for quantification of gene/protein expression (113) (124,125) This gene encodes a protein with pyruvate kinase activity that catalyzes the formation of pyruvate from (126) 15q22 PKM2 Gene The Closure kit cannot replace colonoscopy; it is usually used as an alternative test or screening method for patients who are dissatisfied with other (invasive) methods of screenings [85][86][87][88][89][90] .

Cologuard® test kit
The Cologuard® test kit (registered trademark of Exact Sciences in the U.S) is the first kit which detects colon cancer based on DNA stool sample. This kit identifies colon cancer depending on the DNA markers and blood in stool. The detection system in this kit is based on the identification, amplification and detection of the methylated DNA targets (NDRG4 and BMP3), K-RAS point mutations, and ACTB (a reference gene for quantitative estimation of the total amount of human DNA in each sample) which is performed using the Quantitative Allele-specific Real-time Target and Signal Amplification (QuARTS™) technology. Occult blood in the stool sample is prepared and analyzed for fecal occult blood in a quantitative enzymelinked immunosorbent assay (ELISA) that determines the concentration of hemoglobin in the sample 91 . Recent studies indicate that the sensitivity of the Cologuard kit for diagnosis of colon cancer and large adenomas is 92.3% and 42%, respectively, with specificity of 87% 97-98 . In comparison with FIT, the Cologuard kit's sensitivity is almost two-fold greater (42% vs. 24%) in identifying advanced adenoma 92 . However, this method cannot be a replacement for the diagnostic colonoscopy. The kit should be prescribed and is useful for men and women > 50 years and for any average-risk individuals 91 . Any diet or bowel preparation is not required. In some studies, 13% of people (individuals without cancer or pre-cancer) showed a positive result using this kit, so they were asked to take a colonoscopy 93

PreGen-Plus™ test kit
PreGen-Plus™ test kit (Laboratory Corporation of America, conducted by EXACT Sciences, LabCorp) is an assay used for an early detection of colon cancer and for any moderate-risk individuals. It uses a multitarget assay panel that contains 21 point mutations in K-ras, APC and p53 genes, a microsatellite instability marker (BAT-26) and a proprietary marker, the DNA Integrity Assay (95). Records show that the sensitivity and specificity are almost 94% and 52% for CRC, respectively 96 . PreGen-Plus™ has not been cleared by the FDA 97 .

Pyruvate kinase type M2 (PKM2) test
Pyruvate kinase (PK) is an enzyme which catalyzes the formation of pyruvate from phosphoenolpyruvate (PEP), the rate-limiting step in glycolytic cascade. There are four PK isoforms: L, R, M1, and M2, each expressed in specific tissues 98 . The M2 isoform is a splice variant of M1 and is highly expressed during the embryonic development and tumor formation. It has been demonstrated that tumor cells exclusively express PKM2 99,100 . Another study reported that the expression of PKM2 is elevated in CRC and is also related to later stages and lymph metastasis of CRC 101 . PKM2 is an important enzyme in the metabolism of tumor cells and is a tumor marker in CRC. Therefore, ELISA-based measurement of PKM2 is a new test for the detection of CRC in stool samples 102 . Sensitivity and specificity of this test for a CRC detection is 79% and 81%, respectively. Moreover, positive and negative predictive values are about 74% and 86%, respectively 103 . The efficacy of this test is equal to FOBT. Li et al. have shown that PKM2 test cannot be used alone for the screening of CRC due to the relatively low specificity and low positive predictive value 104,105 .

CONCLUSION
Over the past several years, different methods have been discovered for an early detection of CRC. Invasive methods will likely be replaced by fecal DNA tests in the future. MiRNAs are also promises for an early detection of CRC. However, other critical matters should also be considered, including cost-effectiveness, optimal testing intervals, and strategies for a follow-up evaluation of patients who have shown a positive result on a fecal DNA test.